At present, no effective therapy is available for the treatment of pancreatic ductal adenocarcinoma (PDAC), which continues to be one of the most lethal cancers. In response to the clinical need to develop innovative therapeutic strategies, this proposal will test the therapeutic efficacy of a novel combinatorial immunotherapeutic strategy, which targets not only differentiated PDAC cells, but also PDAC cancer initiating cells (CICs). These cells, identified as ALDHbright cells, have to be eradicated in order to cure a malignant disease, because, according to the cancer stem cell theory, these cells are responsible for disease recurrence and metastases. The tumor antigen selected as a target is the glucose-regulated protein of 94 kDa (Grp94), a member of the heat shock protein 90 (HSP90) family. Grp94 regulates the activation of several signaling pathways which are associated with cell proliferation, survival and migration. To target Grp94, we will take advantage of the unique specificity of the human mAb W9, which we have recently characterized. mAb W9 recognizes an extracellular Grp94 epitope that is selectively expressed on malignant cells, including human PDAC cells and PDAC CICs, but not in normal tissues. mAb W9 markedly inhibits the in vitro proliferation of human PDAC cells, including PDAC CICs, and induces their apoptosis. The anti-proliferative and pro-apoptotic activity of mAb W9 is enhanced by combining it with cyclopamine and 5-FU. Both cyclopamine and 5-FU inhibit the sonic hedgehog signaling pathway, which is aberrantly activated in PDAC, particularly in the stroma and PDAC CICs. To assess the in vivo relevance of these in vitro data, the specific aims of this proposal will test the following hypotheses: i) The expression of the mAb W9 defined Grp94 epitope in primary PDAC tumors is associated with their histopathological characteristics and with poor prognosis; ii) mAb W9, in combination with cyclopamine and 5-FU, is more effective than individual agents, or a combination of two agents, in eradicating PDAC tumors in immunodeficient mice orthotopically grafted with the PDAC cell line MIA PaCa-2; iii) The results obtained with the PDAC cell line MIA PaCa-2 will have potential clinical significance, as they are reproduced in immunodeficient mice orthotopically grafted with PDAC tumors surgically removed from patients. The information derived from the outlined studies in vitro and in animal model systems will provide a useful background to implement a Phase I clinical trial with the mAb W9 in patients with PDAC. The implementation of this trial will be facilitated by Dr. C. Ferrone's access to her patients wit PDAC at Massachusetts General Hospital, Boston, MA, her newly acquired skills in conducting clinical trials, and by the fully human nature of the mAb W9.